Microscopic images of cellular structure in biological samples can reveal important information regarding biological processes and cellular architecture. A correlative approach, which uses both optical microscopy and electron microscopy, produces the most comprehensive results. For example, light microscopy information can be used to identify areas of biological importance and their dynamics within a sample. Then electron microscopy can be used to resolve structural details within those areas after fixation and/or staining.
Images collected with a conventional optical microscope are limited in resolution to about half of the wavelength of the light used. For practical optical microscopy this limit is around 200 nm. Because of this limitation, conventional optical microscopes are said to be diffraction limited. Many techniques exist for improving resolution beyond the diffraction limit. Such techniques are called super-resolution techniques. One particular technique is stochastic optical reconstruction microscopy (STORM). Another technique is photo-activated localization microscopy (PALM). These techniques are used to form an image of a sample using fluorescent markers which can be switched between an “on” state, in which the marker fluoresces, and an “off” state, in which the marker does not fluoresce. STORM typically uses fluorescent organic dyes whereas PALM typically uses fluorescent proteins. The switching between states is realized when the markers enter a dark state after fluorescent emission and are then insensitive to excitation for a period of time. Due to this inactivation, the vast majority of markers are in the dark state at a given time with only a small number emitting fluorescent light. In forming a super-resolution image of a sample, a large series of separate images of the sample are collected to localize each individual marker independent of neighboring markers.
In the separate images, each marker appears as a diffraction-limited point-spread function. A Gaussian fit is applied to each point-spread function, and the marker location is now represented by a point at the center of the Gaussian fit. By sequential imaging and application of this process to each marker, a super-resolution image of the sample is built up, allowing imaging past the diffraction limit. Different colored fluorescent dyes can be imaged simultaneously using, for example, dichroic optics selected to separate the emissions of different markers based on their emission spectra. Using several wavelength channels can allow imaging of several different cellular components simultaneously.
One variation of the PALM is interferometric PALM, or “iPALM.” By arranging multiple lenses, for example one lens above and one lens below the sample, fluorescent light collected can be caused to interfere with itself so as to produce an interference pattern which depends on the difference in the optical path length between the two lens systems. This allows localization in the Z dimension.
Non-superresolution techniques such as confocal imaging also allow for three dimensional fluorescence imaging albeit with reduced resolution. The invention may also be advantageous to correlative microscopy involving these types of optical imaging modalities as well.
Correlative microscopy involves overlaying one or more images created with one imaging technique with one or more images created using another imaging technique. For example, one image may be formed by an optical microscope and another image may be formed by a charged particle beam microscope. In one example, iPALM is used to form an optical image and a scanning electron beam is used to form a series of images, and the images are correlated. The iPALM technique provides localization information about specific regions in a sample, while an image from the electron microscope can show overall characteristics of a sample. This process is especially useful in the imaging of biological samples in which specific proteins or other structures in the biological sample can be chemically functionalized with organic dyes or genetically modified to express fluorescent protein, which can be imaged with iPALM. Correlating iPALM data with data from a charged particle system provides contextual information about the location of the fluorescent marker within the ultrastructure of the sample. Choosing appropriate charged particle preparation and imaging techniques, a three dimensional image can be constructed to give an excellent perspective of where in a sample specific features are located.
In the correlative microscopy example described above, iPALM is used to obtain three-dimensional super-resolution fluorescent images of a sample, first by sequentially localizing an area of interest in an X-Y image plane and rendering a two-dimensional super-resolution image from the molecular coordinates. Simultaneous multiphase interference of light emitted from each molecule is further used to extract a Z axis location, defining a third dimension. The same samples imaged using iPALM are then imaged by a charged particle system. The charged particle system may operate in a cycle in which, for example, a focused ion beam (FIB) removes a few-nanometer-thick layer of sample to expose a new surface that is imaged by SEM. This cycle may repeat numerous times to form a stack of images of ever-deeper layers in the sample.
Correlation of iPALM and electron microscopy (EM) images, however, is limited. Existing methods for correlation involve the use of a planar layer of fiducials at the interface of the sample volume and a supporting substrate. This allows accurate location information in the X-Y plane, but poor localization in the Z-plane. For example, correlation in the two dimensional X-Y plane produces excellent data using the technique as described in U.S. Pat. No. 7,924,432, issued to Hess et al. (“Hess”). In this technique, correlation in the X and Y dimensions are generally straightforward. However, the correlation of the Z plane using the method of Hess relies on interpolation between the top and bottom surfaces of the sectioned sample. This becomes problematic because the sample section can undergo changes due to electron and ion beam-induced distortion as well as changes that can occur in the sample due to sample preparation and insertion into vacuum for charged particle processing.
When biological samples are prepared for charged particle microscopy, physical changes to the sample often result. These physical changes can occur due to the “wet” preparation of a sample. One example of such a preparation is staining the sample with heavy metal stains which are visible in a charged particle system. Physical changes can also result from exposure of a sample to the vacuum environment in the charged particle system. These physical changes degrade the ability to correlate iPALM images with charged particle images of the same sample to obtain valuable information of the sample especially in the Z dimension.
Some attempts have been made to overcome the deficiencies of accurate imaging in the Z dimension. Such attempts include the use of fluorescent markers on the top surface of the sample. However, such attempts do not overcome the deficiencies in data correlation due to deformation of the sample. Another difficulty presented by current methods of using fluorescent markers is the presence of fluorescent dye throughout the sample volume containing the markers. If dye is present throughout the sample volume, typically too much dye is present for accurate localization of the marker using the stochastic iPALM or STORM process, which requires imaging individual single photon emission events. As a result, the brightness of a dye dispersed throughout the sample volume may produce so much fluorescence that it is difficult to accurately locate nearby areas of interest.